Hairpin ribozymes

ABSTRACT

Hairpin ribozyme lacking a substrate moiety, comprising at least six bases in helix 2 and able to base-pair with a separate substrate RNA, wherein the said ribozyme comprises one or more bases 3&#39; of helix 3 able to base-pair with the said substrate RNA to form a helix 5 and wherein the said ribozyme can cleave and/or ligate said separate RNA(s) in trans.

This is a continuation of application Ser. No. 08/321,993 filed Oct. 11,1994, U.S. Pat. No. 5,631,359, hereby incorporated by reference in itstotality (including drawings).

BACKGROUND OF THE INVENTION

This invention relates to hairpin ribozymes.

The following is a brief description of publications concerningribozymes, and in particular, hairpin ribozymes. None are admitted to bethe prior art to the pending claims, and all are incorporated byreference herein.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. Table 1 summarizes some of the characteristics of theseribozymes. In general, enzymatic nucleic acids act by first binding to atarget RNA. Such binding occurs through the target binding portion of aenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cut the target RNA. Strategic cleavage of such atarget RNA will destroy its ability to direct synthesis of an encodedprotein. After an enzymatic nucleic acid has bound and cleaved its RNAtarget, it is released from that RNA to search for another target andcan repeatedly blind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over othertechnologies, such as antisense technology (where a nucleic acidmolecule generally simply binds to a nucleic acid target to block itstranslation) since the concentration of ribozyme necessary to affect atherapeutic treatment is lower than that of an antisenseoligonucleotide. This advantage reflects the ability of the ribozyme toact enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA. In addition, the ribozyme is a highlyspecific inhibitor, with the specificity of inhibition depending notonly on the base pairing mechanism of binding to the target RNA, butalso on the mechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992 Proc. Natl.Acad. Sci. USA, 89, 7305-7309). Thus, the specificity of action of aribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site.

Van Tol et al., 1991 (Virology 180, 23) describe a hairpin ribozymestructure able to circularize. Hisamatsu et al., 1993 (Nucleic AcidsSymp. Ser. 29, 173) describe hairpin ribozymes having a long substratebinding site in helix 1. Berzal-Herranz et al., 1993 (EMBO J. 12,2567)describe essential nucleotides in the hairpin ribozyme. Hampel andTritz, 1989 (Biochemistry 28, 4929) describe a hairpin ribozyme derivedfrom the minus strand of tobacco ringspot virus satellite (-) sTRSV!RNA. Haseloff and Gerlach 1989 (Gene 82, 43) describe sequences requiredfor self-cleavage reactions catalyzed by the (-) sTRSV RNA. Feldstein etal., 1989 (Gene 82, 53) tested various models of trans-cleaving motifsderived from (-) sTRSV RNAs. The hairpin ribozyme can be assembled invarious combinations to catalyze a unimolecular, bimolecular or atrimolecular cleavage/ligation reaction (Berzal-Herranz et al., 1992,Genes & Develop. 6, 129; Chowrira and Burke, 1992 Nucleic Acids Res. 20,2835; Komatsu et al., 1993 Nucleic Acids Res. 21, 185; Komatsu et al.,1994 J. Am. Chem. Soc. 116, 3692). Increasing the length of helix 1 andhelix 4 regions do not affect the catalytic activity of the hairpinribozyme (Hisamatsu et al., 1993 supra; Chowrira and Burke, 1992 supra;Anderson et al., 1994 Nucleic Acids-Res. 22, 1096). For a review ofvarious ribozyme motifs, and hairpin ribozyme in particular, see Ahsenand Schroeder, 1993 Bioessays 15, 299; Cech, 1992 Curr. Opi. Struc. Bio.2, 605; and Hampel et al., 1993 Methods: A Companion to Methods inEnzymology 5, 37.

This invention concerns an improved ribozyme based on the hairpin 30motif described by Hampel and Fritz 1989 supral Feldstein et al., 1989supra; Hampel et al., 1990 Nucleic Acid Res. 18, 299; and Hampel et alEP 0360257.

Hairpin ribozyme-substrate complex comprises of two intermolecularhelices formed between the ribozyme and the target RNA (helix 1 andhelix 2). Length of helix 1 can be varied substantially withouteffecting the catalytic activity of the ribozyme (Hisamatsu et al., 1993supra). However, the length of helix 2 is reported to be sensitive tovariation. The length of helix 2 is normally between 3 and 5 base-pairslong (Hampel & Tritz, 1989 supra; Feldstein et ali 1989 supra; Haseloffand Gerlach, 1989 supra; Hampel et al., 1990 supra; Feldstein et al,1990 Proc. Natl. Acad. Sci. USA 87 2623). Several reports suggest thatmutations within this helix significantly inhibit ribozyme activity(Hampel et al, 1990 supra; Feldstein et al., 1990 supra; Chowrira &Burke, 1991 Biochemistry 30, 8518; Joseph et al., 1993 Genes & Develop.7, 130). It is also believed in the art that the length of helix 2should be between 3 and 5 bp (Hampel et al., 1988 EPO 360 257; Hampel etal., 1993 supra, Cech, 1992 supra; von Ahsen and Schroeder, 1993 supra;Hisamatsu et al., 1993 supra, Anderson et al., 1994 supra).

SUMMARY OF THE INVENTION

This invention features an improved trans-cleaving hairpin ribozyme inwhich a new helix (i.e., a sequence able to form a double-strandedregion with another single-stranded nucleic acid) is provided in theribozyme to base-pair with a 5' region of a separate substrate nucleicacid. This helix is provided at the 3' end of the ribozyme after helix 3as shown in FIG. 6. In addition, at least two extra bases may beprovided in helix 2 and a portion of the substrate corresponding tohelix 2 may be either directly linked to the 5' portion able to hydrogenbond to the 3' end of the hairpin or may have a linker of atleast onebase. By trans-cleaving is meant that the ribozyme is able to act intrans to cleave another RNA molecule which is not covalently linked tothe ribozyme itself. Thus, the ribozyme is not able to act on itself inan intramolecular cleavage reaction.

By "base-pair" is meant a nucleic acid that can form hydrogen bond(s)with other RNA sequence by either traditional Watson-Crick or othernon-traditional types (for example Hoogsteen type) of interactions.

Referring to FIG. 6, there is shown in diagrammatic form the generalstructure of a ribozyme of this invention. Helix 2 (H2) is provided witha least 4 base pairs (ie., n is 1, 2, 3 or 4) and a new helix 5 isprovided of length 2 or more bases (preferably 3-20 bases, ie., m isfrom 1-20 or more). Helix 2 and helix 5 may be covalently linked by oneor more bases (i.e., r is 1 base). Together, these modifications of thehairpin (HP) structure are advantageous in enhancing stability to theribozymeesubstrate complex. Helix 4 may also be extended by 2 or morebase pairs (e.g., 4-10 base pairs) to stabilize the ribozyme structure.In each instances, each N and N' independently is any normal or modifiedbase and each dash represents a potential base-pairing interaction.These nucleotides may be modified at the sugar, base or phosphate.Complete base-pairing is not required in the helixes, but is preferred.Helix 1 and 4 can be of any size (i.e., o and p is each independentlyfrom 0 to any number, e.g., 20) as long as some base-pairing ismaintained. Essential bases are shown as specific bases in thestructure, but those in the art will recognize that one or more may bemodified chemically (abasic, base, sugar and/or phosphate modifications)or replaced with another base without significant effect. Helix 4 can beformed from two separate molecules, i.e., without a connecting loop. Theconnecting loop when present may be a ribonucleotide with or withoutmodifications to its base, sugar or phosphate. "q" is ≧2 bases. Theconnecting loop can also be replaced with a non-nucleotide linkermolecule (see FIG. 17 for more details). H, refers to bases A, U or C. Yrefers to pyrimidine bases. "" refers to a chemical bond.

The increase in length of helix 2 of a hairpin ribozyme (with or withouthelix 5) has several advantages. These include improved stability of theribozyme-target complex in vivo. In addition, an increase in therecognition sequence of the hairpin ribozyme improves the specificity ofthe ribozyme. This also makes possible the targeting of potentialhairpin ribozyme sites that would otherwise be inaccessible due toneighboring secondary structure.

The increase in length of helix 2 of a hairpin ribozyme (with or withouthelix 5) enhances trans-ligation reaction catalyzed by the ribozyme.Transligation reactions catalyzed by the regular hairpin ribozyme (4 bphelix 2) is very inefficient (Komatsu et al, 1993 Nucleic Acids Res. 21,185). This is attributed to weak base-pairing interactions betweensubstrate RNAs and the ribozyme. By increasing the length of helix 2(with or without helix 5) the rate of ligation (in vitro and in vivo)can be enhanced several fold.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings:

FIG. 1 is a diagrammatic representation of the hammerhead ribozyme (Seq.Id. NO. 1) (Seq. Id. NO. 2) domain known in the art. Stem II can be ≧2base-pair long, or can even lack base pairs and consist of a loopregion.

FIG. 2a is a diagrammatic representation of the hammerhead ribozymedomain known in the art; FIG. 2b is a diagrammatic representation of thehammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 3 27,596-600) into a substrate and enzyme portion; FIG. 2c is a similardiagram showing the hammerhead divided by Haseloff and Gerlach (1988,Nature, 334, 585-51) into two portions; and FIG. 2d is a similar diagramshowing the hammerhead divided by Jeffries and Symons (1989, Nucleic.Acids. Res., 17,1371-1371) into two portions.

FIG. 3 is a representation of the general structure of the hairpinribozyme (Seq. Id. NO. 3) (Seq. Id. NO. 4) domain known in the art. H,is A, U or C. Y is U or C. N is A, U, G, or C. N' is the complementarysequence of N. Helix 4 can be ≧2 base-pair long.

FIG. 4 is a representation of the general structure of the hepatitisdelta virus ribozyme (Seq. Id. NO. 5) domain known the art.

FIG. 5 is a representation of the general structure of the VS RNAribozyme (Seq. Id. NO. 6) domain.

FIG. 6 is a diagrammatic representation of a ribozyme of this invention(Seq. Id. NO. 7) (Seq. Id. NO. 8) see above for a complete description).

FIG. 7 is a diagrammatic representation of a Site A Hairpin Ribozyme(Seq. Id. NO. 44) (Seq. Id. NO. 45) (HP-A) showing the proposedsecondary structure of the hairpin ribozyme substrate complex asdescribed in the art (Berzal-Herranz et al, 1993 EMBO. J.12, 2567). Theribozyme has been assembled from two fragments (bimolecular ribozyme;Chowrira and Burke, 1992 Nucleic Acids Res. 20, 2835); #H1 and H2represent intermolecular helix formation between the ribozyme and thesubstrate. H3 and H4 represent intramolecular helix formation within theribozyme (intermolecular helix in the case of bimolecular ribozyme).Left panel (HP-A1) indicates 4 base-paired helix 2 and the right panel(Seq. Id. NO. 44) (Seq. Id. NO. 47) (Seq. Id. NO. 48) (HP-A2) indicates6 base-paired helix 2. Arrow indicates the site of RNA cleavage. All theribozymes discussed herein were chemically synthesized by solid phasesynthesis using RNA phosphoramadite chemistry, unless otherwiseindicated. Those skilled in the art will recognize that these ribozymescould also be made transcriptionally in vitro and in vivo.

FIG. 8 of a graph showing RNA cleavage by hairpin ribozymes targeted tosite A. A plot of fraction of the target RNA uncleaved (fractionuncleaved) as a function of time is shown. HP-A2 (6 bp helix 2) cleavesa 422 target RNA to a greater extent than the HP-A1 (4 bp helix 2).

To make internally-labeled substrate RNA for trans-ribozyme cleavagereactions, a 422 nt region (containing hairpin site A) was synthesizedby PCR using primers that place the T7 RNA promoter upstream of theamplified sequence. Target RNA was transcribed in a standardtranscription buffer in the presence of α-³² P!CTP (Chowrira & Burke,1991 supra). The reaction mixture was treated with 15 units ofribonuclease-free DNasel, extracted with phenol followedchloroform:isoamyl alcohol (25:1), precipitated with isopropanol andwashed with 70% ethanol. The dried pellet was resuspended in 20 μlDEPC-treated water and stored at -20° C.

Unlabeled ribozyme (1 μM) and internally labeled 422 nt substrate RNA(<10 nM) were denatured and renatured separately in a standard cleavagebuffer (containing 50 mM Tris-HCl pH 7.5 and 10 mM MgCl₂) by heating to90° C. for 2 min. and slow cooling to 37° C. for 10 min. The reactionwas initiated by mixing the ribozyme and substrate mixtures andincubating at 37° C. Aliquots of 5 μl were taken at regular timeintervals, quenched by adding an equal volume of 2X formamide gelloading buffer and frozen on dry ice. The samples were resolved on 5%polyacrylamide sequencing gel and results were quantitatively analyzedby radioanalytic imaging of gels with a Phosphorlmager (MolecularDynamics, Sunnyvale, Calif.).

FIG. 9 is the Site B Hairpin Ribozyme (Seq. Id. NO. 9) (Seq. Id. NO. 10)(Seq. Id. NO. 11) (HP-B) showing proposed secondary structure of thehairpin ribozyme.substrate complex. The ribozyme was assembled from twofragments as described above. The nomenclature is the same as above.(Seq. Id. NO. 12) (Seq. Id. NO. 13) (Seq. Id. NO. 14)

FIG. 10 a graph showing RNA cleavage by hairpin ribozymes targeted tosite B. A. plot of fraction of the target RNA uncleaved (fractionuncleaved) as a function of time is shown. HP-B2 (6 bp helix 2) cleavesa 2 KB target RNA to a greater extent than the HP-B1 (4 bp helix 2). Tomake internally-labeled substrate RNA for trans-ribozyme cleavagereactions, a 2 kB region (containing hairpin site B) was synthesized byPCR using primers that place the T7 RNA promoter upstream of theamplified sequence. The cleavage reactions were carried out as describedabove.

FIG. 11 shows a Site C Hairpin Ribozyme (Seq. Id. NO. 16) (Seq. Id. NO.17) (Seq. Id. NO. 18) (HP-C) with the proposed secondary structure ofthe hairpin ribozymeesubstrate complex. The ribozyme was assembled fromtwo fragments as described above (Seq. Id. NO. 18) (Seq. Id. NO. 19)(Seq. Id. NO. 20).

FIG. 12 is a graph showing RNA cleavage by hairpin ribozymes targeted tosite C. The ribozymes were tested at both 20° C. and at 26° C. To makeinternally-labeled substrate RNA for trans-ribozyme cleavage reactions,a 1.9 KB region (containing hairpin site C) was synthesized by PCR usingprimers that place the T7 RNA promoter upstream of the amplifiedsequence. Cleavage reactions were carried out as described above exceptthat 20° C. and at 26° C. temperatures were used.

FIGS. 13 (Seq. Id. NO. 21) (Seq. Id. NO. 22) (Seq. Id. NO. 23) (Seq. Id.NO. 24) and 15 (Seq. Id. NO. 25) (Seq. Id. NO. 26) (Seq. Id. NO. 27)(Seq. Id. NO. 28) show Site D and E hairpin ribozymes (HP-D/E) within aproposed secondary structure of hairpin ribozyme •substrate complex. Twohairpin ribozymes were designed to cleave two distinct sites (sites Dand sites E) within the mouse TNF-α mRNA. Two more ribozymes weresynthesized containing mutations in the catalytic core of the ribozymewhich renders the ribozyme inactive (Berzal-Herranz et al., 1993 EMBO J.12, 2567). To construct these ribozymes, partially overlapping top- andbottom-strand oligonucleotides (˜50 nucleotides) were designed toinclude sequences for the T7 promoter and the hairpin ribozyme. Thesingle-strand portions of annealed oligonucleotides were converted todouble-strands using Sequenase® (U.S. Biochemicals). Transcriptionreactions containing linear double-stranded templates were carried outessentially as described (Milligan & Uhlenbeck, 1989 supra) using the T7mega shortscript kit (Ambion, Austin, Tex.).

FIGS. 14 and 16 show RNA cleavage by HP-D and HP-E in mammalian cellswith noted controls.

FIGS. 17A-17D show various structural modifications of the presentinvention. A) Hairpin ribozyme (Seq. Id. NO. 29) (Seq. Id. NO. 30)lacking helix 5. Nomenclature is same as described under FIG. 6. B)Hairpin ribozyme (Seq. Id. NO. 31) (Seq. Id. NO. 32) lacking helix 4 andhelix 5. Helix 4 is replaced by a nucleotide loop wherein q is ≧2 bases.Nomenclature is same as described under FIG. 6. C) Hairpin ribozyme(Seq. Id. NO. 33) (Seq. Id. NO. 34) (Seq. Id. NO. 35) lacking helix 5.Helix 4 loop is replaced by a linker "L", wherein L is a non-nucleotidelinker molecule (Benseler et al., 1993 J. Am. Chem. Soc. 115, 8483;Jennings et al, WO 94/13688). Nomenclature is same as described underFIG. 6. D) Hairpin ribozyme (Seq. Id. NO. 36) (Seq. Id. NO. 37) (Seq.Id. NO. 38) lacking helix 4 and helix 5. Helix 4 is replaced bynon-nucleotide linker molecule "L" (Benseler et al., 1993 supra;Jennings et al., supra). Nomenclature is same as described under FIG. 6.

FIG. 18 shows Hairpin ribozymes (Seq. Id. NO. 39) (Seq. Id. NO. 40)(Seq. Id. NO. 41) (Seq. Id. NO. 42) containing nucleotide spacer region"s" at the indicated location, wherein s is ≧1 base. Hairpin ribozymescontaining spacer region, can be synthesized as one fragment or can beassembled from multiple fragments. Nomenclature is same as describedunder FIG. 6

TARGET SITES

Targets for useful ribozymes can be determined as disclosed in Draper etal. WO 93/23569, Sullivan et al, WO 94/02595 as well as by Draper etal., "Method and reagent for treatment of arthritic conditions U.S. Ser.No. 08/152,487, filed Nov. 12, 1993, and hereby incorporated byreference herein in totality. Rather than repeat the guidance providedin those documents here, below are provided specific examples, notlimiting to those in the art. Ribozymes to such targets are designed asdescribed in those applications and synthesized to be tested in vitroand in vivo, as also described. Such ribozymes can also be optimized anddelivered as described therein.

Ribozymes are designed to anneal to various sites in the target RNA. Thebinding arms are complementary to the target site sequences describedabove. The ribozymes are chemically synthesized. The method of synthesisused follows the procedure for normal RNA synthesis as described inUsman et al., 1987 J. Am. Chem. Soc., 109, 7845-7854 and in Scaringe etal., 1990 Nucleic Acids Res., 18, 5433-5441 and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5'-end, and phosphoramidites at the 3'-end. The average stepwisecoupling yields were >98%. Hairpin ribozymes are synthesized in twoparts and annealed to reconstruct the active ribozyme (Chowrira andBurke, 1992 Nucleic Acids Res., 20, 2835-2840). All ribozymes aremodified extensively to enhance stability by modification with nucleaseresistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro,2'-o-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17,34). Ribozymes are purified by gel electrophoresis using general methodsor are purified by high pressure liquid chromatography (HPLC; See Usmanet al., Synthesis, deprotection, analysis and purification of RNA andribozymes, filed May, 18, 1994, U.S. Ser. No. 08/245,736 the totality ofwhich is hereby incorporated herein by reference) and are resuspended inwater.

Ribozyme activity can be optimized by chemically synthesizing ribozymeswith modifications that prevent their degradation by serum ribonucleases(see e.g., Eckstein et al., International Publication No. WO 92/07065;Perrault et al., Nature 1990, 344:565; Pieken et al., Science 1991,253:314; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17:334;Usman et al, International Publication No. WO 93/15187; and Rossi etal., International Publication No. WO 91/03162, as well as Usman, N. etal. U.S. patent application Ser. No. 07/829,729, U.S. Pat. No.5,652,094, and Sproat, B. European Patent Application 92110298.4;Ortigao et al., 2 Antisense research and Development; Krist et al.,Abstracts International conference on antisense nucleic acids,Garmisch-Partenkirchen, 1993; Chowrira and Burke, 1992 supra; Chowriraet al., 1993 J. Biol. Chem. 268, 19458, which describe various chemicalmodifications that can be made to the sugar moieties of enzymatic RNAmolecules. All these publications are hereby incorporated by referenceherein.), modifications which enhance their efficacy in cells, andremoval of helix-containing bases to shorten RNA synthesis times andreduce chemical requirements.

Ribozymes are added directly, or can be complexed with cationic lipids,packaged within liposomes, or otherwise delivered to target cells. TheRNA or RNA complexes can be locally administered to relevant tissues exvivo, or in vivo through injection, aerosol inhalation, infusion pump orstent, with or without their incorporation in biopolymers.

Sullivan, et al., supra, describes the general methods for delivery ofenzymatic RNA molecules. Ribozymes may be administered to cells by avariety of methods known to those familiar to the art, including, butnot restricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres. For someindications, ribozymes may be directly delivered ex vivo to cells ortissues with or without the aforementioned vehicles. Alternatively, theRNA/vehicle combination is locally delivered by direct injection or byuse of a catheter, infusion pump or stent. Other routes of deliveryinclude, but are not limited to, intravascular, intramuscular,subcutaneous or joint injection, aerosol inhalation, oral (tablet orpill form), topical, systemic, ocular, intraperitoneal and/orintrathecal delivery. More detailed descriptions of ribozyme deliveryand administration are provided in Sullivan, et al., supra and Draper,et al., supra which have been incorporated by reference herein.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol IlI promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersare also used, providing that the prokaryotic RNA polymerase enzyme isexpressed in the appropriate cells (Elroy-Stein, O. and Moss, B., 1990,Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao, X. and Huang;, L., 1993,Nucleic Acids Res., 21, 2867-72; Lieber, A., et a., 1993, MethodsEnzymol., 217, 47-66; Zhou, Y., et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that ribozymesexpressed from such promoters can function in mammalian cells (e.g.(Kashani-Sabet, M., et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang,J. O., et al., 1992, Proc. Natl. Acad. Sci. U S A, 89, 10802-6; Chen, C.J., et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu, M., et al., 1993,Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L'Huillier, P. J., et al.,1992, EMBO J., 11, 4411-8; Lisziewicz, J., et al, 1993, Proc. Natl.Acad. Sci. U. S. A., 90, 8000-4)). The above ribozyme transcriptionunits can be incorporated into a variety of vectors for introductioninto mammalian cells, including but not restricted to, plasmid DNAvectors, viral DNA vectors (such as adenovirus or adeno-associatedvectors), or viral RNA vectors (such as retroviral, Semliki forestvirus, sindbis virus vectors).

In a preferred embodiment of the invention, a transcription unitexpressing a hairpin ribozyme that cleaves target RNA (e.g., TNF-α mRNA)is inserted into a plasmid DNA vector or an adenovirus oradeno-associated DNA viral vector. Both viral vectors have been used totransfer genes to the lung and both vectors lead to transient geneexpression (Zabner et al., 1993 Cell 75, 207; Carter, 1992 Curr. Opi.Biotech. 3, 533) The adenovirus vector is delivered as recombinantadenoviral particles. DNA may be delivered alone or complexed withvehicles (as described for RNA above). The DNA, DNA/vehicle complexes,or the recombinant adenovirus particles are locally administered to thesite of treatment, e.g., through the use of an injection catheter, stentor infusion pump or are directly added to cells or tissues ex vivo.

In another aspect of the invention, ribozymes that cleave targetmolecules are expressed from transcription units inserted into DNA, RNA,or viral vectors. Preferably, the recombinant vectors capable ofexpressing the ribozymes are locally delivered as described above, andtransiently persist in target cells. Once expressed, the ribozymescleave the target mRNA. The recombinant vectors are preferably DNAplasmids, adenovirus, retroviral or adeno-associated virus vectors.However, other mammalian cell vectors that direct the expression of RNAmay be used for this purpose.

Thus, ribozymes of the present invention that cleave target mRNA andthereby inhibit and/or reduce target activity have many potentialtherapeutic uses, and there are reasonable modes of delivering theribozymes in a number of the possible indications. Development of aneffective ribozyme that inhibits specific function are described in theart.

By "inhibit" is meant that the activity or level of target RNA isreduced below that observed in the absence of the ribozyme, andpreferably is below that level observed in the presence of an inactiveRNA molecule able to bind to the same site on the RNA, but unable tocleave that RNA.

By "vectors" is meant any nucleic acid--and/or viral-based techniqueused to deliver a desired nucleic acid.

Diagnostic Uses

Ribozymes of this invention may be used as diagnostic tools to examinegenetic drift and mutations within diseased cells, or to detect specificRNA molecules, such as virus RNA. The close relationship betweenribozyme activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple ribozymes described in this invention, one may map nucleotidechanges which are important to RNA structure and function in vitro, aswell as in cells and tissues. Cleavage of target RNAs with ribozymes maybe used to inhibit gene expression and define the role (essentially) ofspecified gene products in the progression of disease. In this manner,other genetic targets may be defined as important mediators of thedisease. These experiments will lead to better treatment of the diseaseprogression by affording the possibility of combinational therapies(e.g., multiple ribozymes targeted to different genes, ribozymes coupledwith known small molecule inhibitors, or intermittent treatment withcombinations of ribozymes and/or other chemical or biologicalmolecules). Other in vitro uses of ribozymes of this invention are wellknown in the art, and include detection of the presence of mRNAassociated with a related condition. Such RNA is detected by determiningthe presence of a cleavage product after treatment with a ribozyme usingstandard methodology.

In a specific example, ribozymes which can cleave only wild-type ormutant forms of the target RNA are used for the assay. The firstribozyme is used to identify wild-type RNA present in the sample and thesecond ribozyme will be used to identify mutant RNA in the sample. Asreaction controls, synthetic substrates of both wild-type and mutant RNAwill be cleaved by both ribozymes to demonstrate the relative ribozymeefficiencies in the reactions and the absence of cleavage of the"non-targeted" RNA species. The cleavage products from the syntheticsubstrates will also serve to generate size markers for the analysis ofwild-type and mutant RNAs in the sample population. Thus each analysiswill require two ribozymes, two substrates and one unknown sample whichwill be combined into six reactions. The presence of cleavage productswill be determined using an RNAse protection assay so that full-lengthand cleavage fragments of each RNA can be analyzed in one lane of apolyacrylamide gel. It is not absolutely required to quantify theresults to gain insight into the expression of mutant RNAs and putativerisk of the desired phenotypic changes in target cells. The expressionof mRNA whose protein product is implicated in the development of thephenotype is adequate to establish risk. If probes of comparablespecific activity are used for both transcripts, then a qualitativecomparison of RNA levels will be adequate and will decrease the cost ofthe initial diagnosis. Higher mutant form to wild-type ratios will becorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

EXAMPLES

Results of experiments suggest that the length of H2 can be 6 bp withoutsignificantly reducing the activity of the hairpin ribozyme. The H2 armlength variation does not appear to be sequence dependent. HP ribozymeswith 6 bp H2 have been designed against five different target RNAs andall five ribozymes efficiently cleaved their cognate target RNA.Additionally, two of these ribozymes were able to successfully inhibitgene expression (e.g., TNF-α) in mammalian cells. Results of theseexperiments are shown below.

HP ribozymes with 7 and 8 bp H2 are also capable of cleaving target RNAin a sequence-specific manner, however, the rate of the cleavagereaction is lower than those catalyzed by HP ribozymes with 6 bp H2.

Example 1

4 and 6 base pair H2

Referring to FIGS. 7-12, HP ribozymes were synthesized as describedabove and tested for activity. Surprisingly, those with 6 base pairs inH2 were still as active as those with 4 base pairs.

Example 2

TNF α ribozymes

Referring to FIGS. 13-16, hairpin ribozymes of this invention weresynthesized as described, and tested in a mammalian cell system asdescribed below.

Macrophage isolation. To produce responsive macrophages 1 ml of sterilefluid thioglycollate broth (Difco, Detroit, Mich.) was injected i.p.into 6 week old female C57bl/6NCRmice 3 days before peritoneal lavage.Mice were maintained as specific pathogen free in autoclaved cages in alaminar flow hood and given sterilized water to minimize "spontaneous"activation of macrophages. The resulting peritoneal exudate cells (PEC)were obtained by lavage using Hanks balanced salt solution (HBSS),plated at 2.5×10⁵ /well in 96 well plates (Costar, Cambridge, Mass.)with Eagles minimal essential medium (EMEM) with 10% heat inactivatedfetal bovine serum. After adhering for 2 hours the wells were washed toremove non adherent cells. The resulting cultures were 97% macrophagesas determined by morphology and staining for non-specific esterase.

Transfection of ribozymes into macrophages: The ribozymes were dilutedto 2X final concentration, mixed with an equal volume of 11 nMlipofectamine (Life Technologies, Gaithersburg, Md.), and vortexed. 100ml of lipid:ribozyme complex was then added directly to the cells,followed immediately by 10 ml fetal bovine serum. Three hours afterribozyme addition 100 ml of 1 mg/ml bacterial lipopolysaccaride (LPS)was added to each well to stimulate TNF production.

Quantitation of TNF-α in mouse macrophages: Supernatants were sampled at0, 2, 4, 8, and 24 hours post LPS stimulation and stored at -70° C.until quantitation, which was done by a TNF-α specific ELISA. ELISAplates were coated with rabbit anti-mouse TNF-a serum at 1;1000 dilution(Genzyme) followed by blocking with blotto and incubation with TNF-αcontaining supernatants. TNF-α was then detected using a murine TNF-αspecific hamster monoclonal antibody (Genzyme). The ELISA was developedwith goat anti-hamster IgG coupled to alkaline phosphatase.

Assessment of reagent toxicity: Following ribozyme/lipid treatment ofmacrophages and harvesting of supernatants viability of the cells wasassessed by incubation of the cells with 5 mg/ml of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).This compound is reduced by the mitochondrial dehydrogenases, theactivity of which correlates well with cell viability. After 12 hoursthe absorbance of reduced MTT is measured at 585 nm.

Inhibition of TNF-α expression by hairpin ribozymes in mousemacrophages: As shown in FIGS. 14 and 16, expression of TNF-α issignificantly inhibited by both active hairpin ribozymes HP-D and HP-E.

The inhibition of TNF-α expression appears to be dependent on thecatalytic activity of the ribozyme, because, catalytically inactivehairpin ribozyme (HP-D dead) does not show appreciable inhibition. HP-E(inactive) ribozyme does show some inhibition of TNF-α expression at 162nM ribozyme concentration. This inhibition may be attributed to someantisense effect.

Other embodiments are within the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 48                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "H"stands for C, A or U.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      NNNNUHNNNNN                                                                   (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      NNNNNCUGANGAGNNNNNNCGAAANNNN                                                  (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "Y"is stands for U or C.                                           The letter "H"stands for A, U or C.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      NNNYNGHYNNNNNN14                                                              (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       NNNNNNNGAAGNNNNNNNNNNAAACANNNNNNNNNNNACAUUACNNNNNN50                          (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 85 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      UGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCCGAGGGGACCG60                UCCCCUCGGUAAUGGCGAAUGGGAC85                                                   (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 176 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GGGAAAGCUUGCGAAGGGCGUCGUCGCCCCGAGCGGUAGUAAGCAGGGAACUCACCUCCA60                AUUUCAGUACUGAAAUUGUCGUAGCAGUUGACUACUGUUAUGUGAUUGGUAGAGGCUAAG120               UGACGGUAUUGGCGUAAGUCAGUAUUGCAGCACAGCACAAGCCCGCUUGCGAGAAU176                   (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "Y"is stands for U or C.                                           The letter "H"stands for A, U or C.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      NNNNNNNYNGHYNNN15                                                             (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "H"stands for C, A or U.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      NNNNGAAGNNNNNNNNNNNAAAHANNNNNNNACAUUACNNNNNNNNN47                             (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      AUACGGUCCCCUGA14                                                              (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     UCAGGGAGAAGUAUACCAGAGAAACACACGCG32                                            (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     CGCGUGGUACAUUACCUGGUA21                                                       (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     AAAUACGGUCCCCUGA16                                                            (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     UCAGGGAGAAGUAUUUACCAGAGAAACACACGCG34                                          (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     CGCGUGGUACAUUACCUGGUA21                                                       (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     CCGCCGUCUCCACCAA16                                                            (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     UUGGUGGAAGAAGCGGACCAGAGAAACACACGCG34                                          (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     CGCGUGGUACAUUACCUGGUA21                                                       (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     GUCCGCCGUCUCCACCAA18                                                          (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     UUGGUGGAAGAAGCGGACACCAGAGAAACACACGCG36                                        (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     CGCGTUGGUACAUUACCUGGUA22                                                      (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     CUGAGCUGUCCCCACCUG18                                                          (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                     CAGGUGGGAGAAGCUCAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA54                      (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                     CUGAGCUGUCCCCACCUG18                                                          (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                     CAGGUGGGAUAAGCUCAGACCAGAGAAAGACACGUUGUGGUACAUUACCUGGUA54                      (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                     GGAAGCUGUCUUCAGACA18                                                          (2) INFORMATION FOR SEQ ID NO: 26:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                     UGUCUGAAAGAAGCUUCCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA54                      (2) INFORMATION FOR SEQ ID NO: 27:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                     GGAAGCUGUCUUCAGACA18                                                          (2) INFORMATION FOR SEQ ID NO: 28:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                     UGUCUGAAAUAAGCUUCCACCAGAGAAAGACACGUUGUGGUACAUUACCUGGUA54                      (2) INFORMATION FOR SEQ ID NO: 29:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                     NNNYNGHYNNN11                                                                 (2) INFORMATION FOR SEQ ID NO: 30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "H"stands for C, A or U.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                     NNNNGAAGNNNNNNNNNNAAAHANNNNNNNACAUUACNNNNNN43                                 (2) INFORMATION FOR SEQ ID NO: 31:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "Y"is stands for U or C.                                           The letter "H"stands for A, U or C.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                     NNNYNGHYNNN11                                                                 (2) INFORMATION FOR SEQ ID NO: 32:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "H"stands for C, A or U.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                     NNNNGAAGNNNNNNNNNNAAAHANNNACAUUACNNNNNN39                                     (2) INFORMATION FOR SEQ ID NO: 33:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "Y"is stands for U or C.                                           The letter "H"stands for A, U or C.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                     NNNYNGHYNNN11                                                                 (2) INFORMATION FOR SEQ ID NO: 34:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "H"stands for C, A or U.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                     NNNNGAAGNNNNNNNNNNAAAHANN25                                                   (2) INFORMATION FOR SEQ ID NO: 35:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                     NNNNACAUUACNNNNNN17                                                           (2) INFORMATION FOR SEQ ID NO: 36:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "Y"is stands for U or C.                                           The letter "H"stands for A, U or C.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                     NNNYNGHYNNN11                                                                 (2) INFORMATION FOR SEQ ID NO: 37:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "H"stands for C, A or U.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                     NNNNGAAGNNNNNNNNNNAAAHA23                                                     (2) INFORMATION FOR SEQ ID NO: 38:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                     NNACAUUACNNNNNN15                                                             (2) INFORMATION FOR SEQ ID NO: 39:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "Y"is stands for U or C.                                           The letter "H"stands for A, U or C.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                     NNNNNNNYNGHYNNN15                                                             (2) INFORMATION FOR SEQ ID NO: 40:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "H"stands for C, A or U.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                     NNNNGAAGNNNNNNNNNNNAAAHANNNNNNNACAUUACNNNNNNNNN47                             (2) INFORMATION FOR SEQ ID NO: 41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "Y"is stands for U or C.                                           The letter "H"stands for A, U or C.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                     NNNYNGHYNNN11                                                                 (2) INFORMATION FOR SEQ ID NO: 42:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "H"stands for C, A or U.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:                                     NNNNGAAGNNNNNNNNNNNAAAHANNNNNNNACAUUACNNNNNNN45                               (2) INFORMATION FOR SEQ ID NO: 43:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:                                     CUGUAGUCCAGGAAUAUGGC20                                                        (2) INFORMATION FOR SEQ ID NO: 44:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:                                     GCCAUAUUCCUGAGAAGCAGACCAGAGAAACACACGCG38                                      (2) INFORMATION FOR SEQ ID NO: 45:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:                                     CGCGUGGUACAUUACCUGGUA21                                                       (2) INFORMATION FOR SEQ ID NO: 46:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:                                     GACUGUAGUCCAGGAAUAUGGC22                                                      (2) INFORMATION FOR SEQ ID NO: 47:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:                                     GCCAUAUUCCUGAGAAGCAGUCACCAGAGAAACACACGCG40                                    (2) INFORMATION FOR SEQ ID NO: 48:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:                                     CGCGUGGUACAUUACCUGGUA21                                                       __________________________________________________________________________

We claim:
 1. A hairpin ribozyme comprising ribonucleotides which cleavesa separate RNA molecule, wherein said hairpin ribozyme comprises:a helix1 region comprising greater than or equal to three nucleotidesinclusive, wherein said helix 1 region base-pairs with said separate RNAmolecule; a helix 2 region comprising between six and eight nucleotidesinclusive, wherein said helix 2 region base-pairs with said separate RNAmolecule; a helix 3 region comprising nucleotides which form at leastfour base-pairs; and a helix 4 region comprising nucleotides which format least two base-pairs.
 2. The hairpin ribozyme of claim 1, whereinsaid ribozyme comprises six nucleotides in the helix 2 region.
 3. Theribozyme of claim 1, having the structure: ##STR1## wherein each N andN' is independently any nucleotide; each A, U, G and C, is adenosine,uridine, guanosine and cytidine respectively and each Y is a pyrimidinenucleotide;each dash may represent a base-pair; p is 1-20, q is 2-20when present, o is 1-20, n is 3-5; and the lines at the 3' and the5'-ends of the ribozyme are meant to indicate additional nucleotideswhich may be present or absent.
 4. The ribozyme of claim 1 having thestructure: ##STR2## wherein each N and N' is independently anynucleotide; each A, U, G and C, is adenosine, uridine, guanosine andcytidine respectively and each Y is a pyrimidine nucleotide;each dashmay represent a base-pair; p is 1-20, o is 1-20, n is 3-5; L is anon-nucleotide linker; and the lines at the 3' and the 5'-ends of theribozyme are meant to indicate additional nucleotides which may bepresent or absent.
 5. The hairpin ribozyme of claim 1, wherein saidhairpin ribozyme comprises at least one sugar modification.
 6. Thehairpin ribozyme of claim 1, wherein said hairpin ribozyme comprises atleast one nucleotide base modification.
 7. The hairpin ribozyme of claim1, wherein said hairpin ribozyme comprises at least one phosphatebackbone modification.
 8. The hairpin ribozyme of claim 1, wherein saidhairpin ribozyme is chemically synthesized.
 9. The hairpin ribozyme ofclaim 1, wherein said cleavage of said separate RNA molecule occurs inthe presence of Mg²⁺.
 10. The hairpin ribozyme of claim 1, wherein saidhairpin ribozyme cleaves a predetermined phosphodiester linkage in saidseparate RNA molecule, wherein said predetermined phosphodiester linkageis immediately 5' of a GHY sequence, wherein G is guanosine, H isadenosine, cytidine or uridine and Y is a pyrimidine nucleotide.
 11. Thehairpin ribozyme of claim 1, wherein said separate RNA molecule isencoded by tumor necrosis factor-alpha gene.
 12. The hairpin ribozyme ofany of claims 11, 2, 3, or 4, wherein said ribozyme ligates RNAmolecules in trans.
 13. A cell including the hairpin ribozyme of any ofclaims 1, 2, 3 or
 4. 14. An expression vector comprising a nucleic acidsequence encoding the hairpin ribozyme of any of claims 1, 2, 3, or 4,in a manner which allows expression of that ribozyme.
 15. The hairpinribozyme of claims 3 or 4, wherein said Y is a uridine or a cytidine.16. The hairpin ribozyme of claims 3 or 4, wherein said p is 2 or 5nucleotides.
 17. The hairpin ribozyme of claims 3 or 4 wherein said o isselected from the group consisting of 4, 6 and 10 nucleotides.
 18. Thehairpin ribozyme of claim 5, wherein said sugar modification is selectedfrom the group consisting of 2'-amino, 2'-C-allyl, 2'-flouro,2'-O-methyl and 2'-H modifications.
 19. The hairpin ribozyme of claim10, wherein said GHY sequence is GUC, wherein G is guanosine, U isuridine and C is cytidine.
 20. The cell of claim 13, wherein said cellis a mammalian cell.
 21. A cell including an expression vector of claim14.
 22. The expression vector of claim 14, wherein said nucleic acidsequence is functionally linked to a RNA polymerase I, or RNA polymeraseII or RNA polymerase III promoter sequence.
 23. The expression vector ofclaim 14, wherein said expression vector is a DNA vector.
 24. Theexpression vector of claim 14, wherein said expression vector is a RNAvector.
 25. The cell of claim 21, wherein said cell is a mammalian cell.26. The expression vector of claim 23, wherein said DNA vector isselected from the group consisting of plasmid DNA, adenovirus andadeno-associated virus vectors.
 27. The expression vector of claim 24,wherein said RNA vector is selected from the group consisting ofretrovirus, semlikiforest virus and sindbis virus vectors.